Autolyzed yeast and sodium butyrate supplemented alone to diets promoted improvements in performance, intestinal health and nutrient transporter in weaned piglets

This study investigated the effects of supplemental nucleotides, autolyzed yeast (Saccharomyces cerevisiae), and sodium butyrate in diets for nursery pigs on growth performance, diarrhea incidence, blood profile, intestinal morphology, mRNA expression of nutrient transporters, inflammatory markers, antioxidant profile, and tight junction proteins in the small intestine. One hundred eighty 21-day-old pigs (5.17 ± 0.57 kg) were assigned in a randomized block design to 1 of 4 dietary treatments: (1) CON: control, basal diet, (2) NUC: CON + nucleotides, (3) YSC: CON + lysed yeast S. cerevisiae, (4) ASB: CON + acidifier sodium butyrate. Pigs were fed for 24 days, phase 1 (21–32 days) and 2 (32–45 days). During phase 1, YSC and ASB improved average daily gain (ADG) and feed conversion (FC) compared with CON. At the overall period, ASB improved ADG and YSC improved FC compared with CON. The NUC diet did not affect growth performance. The ASB increased ileal villus height compared to CON. The YSC and ASB reduced the number of Peyer’s patches in the ileum compared with CON. The YSC increased mRNA expression of nutrient transporters (SMCT2, MCT1, and PepT1), tight junction proteins (OCL and ZO-1), antioxidants (GPX), and IL1-β in the jejunum compared with CON. The ASB increased mRNA expression of nutrient transporters (SGLT1 and MCT1), tight junction proteins (OCL and ZO-1), and antioxidants (GPX and SOD) compared with CON. In conclusion, autolyzed yeast and sodium butyrate promoted growth performance by improving the integrity of the intestinal barrier, the mRNA expression of nutrient transporters, and antioxidant enzymes in the jejunum of nursery pigs whereas supplementation of nucleotides did not show such effects.


Growth performance
During phase 1 (21-32 days), pigs fed YSC or ASB diets showed (P < 0.05) improvements in ADG, FC, and BW compared with pigs fed CON diet; however, ADFI was not affected by dietary treatments (Table 1).In phase 2 (32-45 days), pigs fed ASB diet had higher (P < 0.05) BW compared to pigs fed CON diet.In addition, there was a trend to increase the BW (P = 0.095) in pigs fed YSC diet compared to those fed CON diet.Pigs fed NUC diet tended to present lower ADG (P = 0.080).However, FC was not affected by dietary treatments.During the overall period, ADG was higher (P < 0.05) in pigs fed ASB diet than those with CON diet.In addition, FC was better (P < 0.05), and a trend was observed to improve ADG (P = 0.095) in pigs fed YSC diet compared with pigs on CON diet.Moreover, ADFI was not affected by dietary treatments.

Diarrhea incidence
There was a trend to reduce the diarrhea incidence (P = 0.098) in pigs fed NUC diet and improved fecal score (P = 0.090) in pigs fed YSC diet than pigs fed CON diet in the phase 1 (Table 2).In phase 2, there was a trend to reduce the diarrhea incidence (P = 0.089) and improved fecal score (P = 0.091) in pigs fed YSC diet than in those fed CON diet.

Blood profile
There was no effect of dietary treatments on IgG, creatinine, and urea concentrations (Table 3).

Intestinal morphology
Dietary treatments had no effects on villus height, crypt depth, villus:crypt ratio, and proportion of goblet cells in the duodenum and jejunum (Table 4).In the ileum, the dietary treatments had no effects on crypt depth and proportion of goblet cells.However, villus height was increased (P < 0.05) and villus:crypt ratio trended to increase (P = 0.066) in pigs fed ASB diet than pigs fed CON diet.In addition, the number of Peyer's patches in pigs fed YSC or ASB diets was lower (P < 0.05) compared to pigs that received CON diet.There was a trend towards an increase (P = 0.071) villus height and reduce (P = 0.078) the number of Peyer's patches in pigs fed NUC diet than those fed the CON diet.

mRNA relative abundance of nutrient transporters
In the jejunum, there was no effect of dietary treatments on the mRNA expression of GLUT2, EAAC1, and y + LAT1 (Fig. 1).However, mRNA expression of SMCT2 and PepT1 was higher (P < 0.05) in pigs fed YSC diet compared to pigs fed CON diet.The mRNA expression of MCT1 in pigs fed ASB and YSC diets was higher Table 1.Growth performance of piglets fed diets supplemented or not with feed additives (n = 9 pens replicates per dietary treatment and 5 piglets per pen as an experimental unit).a Average daily feed intake (ADFI, g/day), average daily weight gain (ADG, g/day), feed conversion ratio (FC, g:g).b Dietary treatment: phase 1 (21-32 days) and phase 2 (32-45 days), respectively: (1) CON: control, basal diet, (2) NUC: CON + 1 g/kg and 0.5 g/kg of nucleotides, (3) YSC: CON + 20 g/kg and 10 g/kg of lysed yeast S. cerevisiae, (4) ASB: CON + 1.5 g/kg and 1 g/kg of acidifier sodium butyrate.c Pooled standard error of the mean.www.nature.com/scientificreports/(P < 0.05) than in those fed CON diet.In addition, SGLT1 mRNA expression in pigs fed ASB diet was higher (P < 0.05), and YSC diet (P = 0.055) trended upwards compared to pigs fed CON diet.

mRNA relative abundance of inflammatory and antioxidants markers, and tight junction proteins
There was no effect of dietary treatments on the mRNA expression of CAT, IFN-γ, and IL-10 (Fig. 2).However, mRNA expression of GPX, OCL, and ZO-1 was higher (P < 0.05) in pigs fed YSC or ASB diets compared to pigs fed CON diet.The mRNA expression of SOD in pigs fed ASB diet, and IL1-β in pigs fed YSC diet was higher (P < 0.05) than in those fed CON diet.In addition, TNF-α mRNA expression in pigs fed YSC diet (P = 0.082) and ASB diet (P = 0.061) trended upwards compared to pigs fed CON diet.There was a downward trend (P = 0.058) in IL1-β mRNA expression of pigs fed NUC diet than pigs that received CON diet.

Discussion
The hypothesis of the current experiment was that the dietary supplementation of purified nucleotide, S. cerevisiae yeast, or encapsulated acidifier sodium butyrate would improve the growth performance of weaned piglets and beneficially affect gut parameters.The present results showed that feeding nursery pigs with YSC or ASB diets increased growth performance and improved gut health, whereas no improvements were observed feeding the NUC diet.
Although nucleotides can be synthesized from other precursors in pigs' diets, in the post-weaning a stressful and limited nutrient intake period, nucleotides could be considered an essential nutrient 6 .However, in the present study, performance was not influenced by NUC diet, while others have reported positive effects of nucleotide supplementation on piglet growth performance 26,27 and feed intake 5 .The NUC diets correspond to 100 mg nucleotides/kg diet in phase 1 and 75 mg nucleotides/kg in phase 2 in a 24-day trial and were fed purified.On the other side, Superchi et al. 26 used a nucleotide yeast-derived source that also contains viable cells, cell wall components, inositol, and functional amino acids.Weaver and Kim 27 demonstrated improved growth performance with up to 1000 mg nucleotides/kg during a 28-day trial.Working with different levels of nucleotides (0, 50, 150, 250, and 500 mg/kg) in a 20-day trial with one pig per pen, Jang and Kim 5 only found significant improvements in the feed intake, and when feeding the lower supplementation levels (50 and 150 mg/kg).Thus, www.nature.com/scientificreports/ the discrepancies in growth performance results between studies are justified due to the different sources, the different dosages used, and administration time.
Regarding the YSC diet, the improved growth performance results are attributed to the improvements in intestinal health promoted by the supplementation of S. cerevisiae yeast.This additive contains high amounts of highly digestible protein, essential amino acids, nucleotides, mannanoligosaccharides, and β-glucans 15 .These components present in yeast may have anti-inflammatory properties to reduce intestinal inflammation and, consequently, minimize diarrheal disorders (as observed in the current study) and nutrient malabsorption 1 .According to Kogan and Kocher 19 , β-glucans are capable of blocking fimbriae of pathogenic bacteria and preventing their adhesion to the epithelium of the intestinal mucosa, acting to prevent or eliminate infection.Yeast-based additives support the immune system of piglets by modulating the intestinal microbiota 28 , which may contribute to improve growth performance 29 .
An increase in nutrient digestibility as reported by Barbosa et al. 30 and Boontiam et al. 8 is another mechanism by which yeast supplementation in nursery pigs' diets can improve the growth performance.Nutrient transporters are proteins expressed in the apical membrane of intestinal cells that absorb nutrients and, therefore, it is possible to improve digestibility by increasing the expression of these transporters 31 .This is supported by the results of our study, which showed increased mRNA expression of nutrient transporters (SGLT1, SMCT2, MCT1, and PepT1) in the jejunum of pigs.The SGLT1 is responsible for glucose absorption, while PepT1 acts on peptide absorption.The increase in the expression of these transporters suggested that there was greater availability of glucose and amino acids at the cellular level, in agreement with the findings by Clarke et al. 31 .Similarly, the increased expression of SMCT2 and MCT1 indicated a greater availability of monocarboxylates (e.g.lactate, short-chain fatty acids, and ketone bodies) 32 , which represent substrates for maintaining the energetic state in cells like the enterocytes.
Regarding the ASB diet, the improvement in growth performance is supported by the increase in SGLT1 and MCT1 mRNA expression in the jejunum of nursery pigs.The MCT1 is expressed in both the small and large intestines, and its function is to transport butyrate into the cell 33 .Although, in the small intestine, microbial butyrate formation is low or absent 34 , the addition of butyrate to diets exerts trophic effects 35 and stimulates the secretion of digestive enzymes 21 , and this can result in more efficient digestion and absorption of dietary nutrients, leading to improved performance.Furthermore, butyrate is a source of energy for enterocytes and additionally has an antiapoptotic effect 33 .Therefore, greater MCT1 and SGLT1 expression may enhance the absorption of nutrients and energy to cope with post-weaning stress 36 .In the present study, sodium butyrate was added in encapsulated form (composed of a vegetable fat-based coating material) to be enzymatically broken down by lipase secreted in the duodenum, as mentioned by Maito et al. 10 .According to Tugnoli et al. 25 , the encapsulation process provides protection that allows a gradual release of the acidifier along the length of the gastrointestinal tract, reducing the dissociation in the stomach and maintaining their efficacy in jejunum and ileum.
The weaning transition promotes physiological changes in the structural and functional aspects of the intestine, causing villous atrophy and increased crypt depth, which in turn reduces the small intestine capacity to absorb nutrients 3 .In the present study, villus height and villus:crypt ratio in the ileum were higher in pigs fed the ASB diet, indicating improvement in intestinal morphology.Moreover, a greater abundance of jejunal mucosal barrier function-related genes (OCL and ZO-1) was observed in pigs fed the ASB diet.These results indicated that ASB diet was efficient in the structural maintenance of the small intestine, as suggested by others 35 .The gradual release of sodium butyrate throughout the intestinal tract seems to be critical in achieving the expected target 9 , allowing the additive to affect different portions of the intestine such as jejunum and ileum.According to Guilloteau et al. 33 , supplementation with sodium butyrate stimulates the proliferation of epithelial cells, resulting in a greater absorption surface and preservation of villi length, reflecting the improvements observed in growth performance of pigs.
A greater abundance of jejunal OCL and ZO-1 mRNA expression was also observed in pigs fed the YSC.According to Rose et al. 37 , OCL and ZO-1 are classified as intestinal junction proteins with a role in regulating epithelial permeability.Decreased intestinal permeability can be correlated with reduced oxidative stress, because this results in attenuation of damage to the intestinal mucosal barrier 14 .Oxidative stress is a physiological stage in which antioxidant defense is inadequate to detoxify the reactive oxygen species, this oxidative process damages essential biomolecules, leading to reduced growth performance 29 .The GPX and SOD as the key enzymes of the antioxidant system play a crucial role in eliminating free radicals, reducing oxidative damage, and maintaining cell structure is well known 38 .In our study, in addition to the increase in the expression of tight junction proteins, pigs fed the ASB and YSC diet had greater expression of GPX and SOD mRNA.These results suggested that nursery pigs fed with additives had greater antioxidant capacity than those fed the CON diet, demonstrating that there was an improvement in the intestinal redox state.
The impacts of weaning stress are not only limited to intestinal barrier function and oxidative stress, but an increase in the activation of the immune system in weaned piglets is also observed 3 .An unregulated enhanced immune response may trigger a negative effect on other metabolic processes and as a result impair growth performance 39 .Peyer's patches are the major organized lymphoid structures involved in the induction of mucosal immune responses in the intestine 40 .In the present study, a reduction in Peyer's patch counts was observed in the ileum of pigs fed YSC or ASB diet.This suggested that there was less induction of the mucosal immune system in pigs that received YSC and ASB diets and, consequently, less stimulus to the immune system because Peyer's patches can be considered as immunological sensors of the intestine 41 .These results suggested that both yeast and sodium butyrate could help enhance the small intestine epithelial barrier, antioxidant capacity, and immune system.Thus, the enhancement of overall gut health helps explain the improvement in the growth performance of pigs fed YSC and ASB diets.
Regarding the production of pro-inflammatory cytokines, it was found that pigs fed the YSC diet showed an increase in IL1-β mRNA expression and a tendency to increase TNF-α, while piglets fed the ASB diet showed www.nature.com/scientificreports/ a tendency to increase TNF-α mRNA expression.On the other hand, pigs fed the NUC diet had a tendency to reduce IL1-β mRNA expression.Nucleotides, yeast, and acidifier-based additives can improve pig immune responses in the post-weaning period 16 .These additives can activate immune cells, including macrophages, which produce IL-1β as part of the immune response 42,43 .The results of the present study indicated a sustained condition of immune response, because in some cases, a controlled and transient increase in IL-1β may be part of a healthy immune response to support the ability of pigs to fight infections or maintain intestinal health 18 .According to Grimble 39 , it is considered beneficial the presence of cytokines (e.g.IL-1β and TNF-α) in adequate concentrations during an inflammatory response to infection.It is important to avoid overstimulation of the immune system, as greater expression of pro-inflammatory cytokines can trigger pathological responses in inflammatory conditions 44 .However, in a previous study, it was observed that TNF-α increases the expression of specific anti-apoptotic proteins, as well as triggers the expression of the survival gene BCL2A1 (not evaluated in the current study) in the intestine of weaned piglets 45 .Also, the β-glucans present in yeast are recognized by specific receptors (pattern recognition receptors) on immune cells, such as macrophages and neutrophils.
In particular, they are recognized by the dectin-1 receptor 46 .Once β-glucans bind to these receptors, they can trigger an immune response.Upon recognition of β-glucans, immune cells can produce pro-inflammatory cytokines, such as TNF-α, and IL-1β 47 .Collectively, although these cytokines play a central role in initiating and magnifying the inflammatory response, they did not negatively affect the biological response of nursery pigs fed YSC and ASB diets.

Conclusions
Based on the evaluated criteria, dietary supplementation of autolyzed yeast S. cerevisiae or sodium butyrate promotes better growth performance by improving the integrity of the intestinal barrier, the mRNA expression of nutrient transporters and antioxidant enzymes in the jejunum of nursery pigs, but without major differences in intestinal morphology in those fed with S. cerevisiae yeast.Furthermore, none of the dietary treatments promoted changes in the observed blood metabolites, but a diet containing S. cerevisiae yeast or sodium butyrate provided a sustained immune response in the jejunum of nursery pigs.On the other hand, dietary nucleotide supplementation did not improve growth performance and gut health.

Material and methods
The experimental protocol follows the ethical principles in animal research (CONCEA, 2016) and was approved by the Ethical Committee on Animal Use of Universidade Federal de Viçosa, under protocol no.0110/2022.All methods were carried out in accordance with relevant guidelines and regulations.All methods were reported in accordance with ARRIVE guidelines (https:// arriv eguid elines.org/ arrive-guide lines).

Animals, experimental design, housing, and diets
The experiment was conducted on a commercial farm in the municipality of Santo Antônio do Grama, MG, Brazil.A total of 180 piglets [PIC 337 (Large White × Landrace × Duroc × Pietrain) × DB 90 (Large White × Landrace)] castrated males and females, weaned at 21 day-old and with 5.17 ± 0.57 kg BW were used in a 24-day trial.Piglets were assigned to a randomized block design based on their initial BW (light 4.7 ± 0.25 kg and heavy 5.6 ± 0.34 kg) into 4 dietary treatments and 9 pens replicates (5 pigs/pen).
The experiment was conducted only in one series, without an adaptation period.The pigs were housed in suspended pens (1.75 × 1.00 m, 0.35 m 2 /pig), with a plastic floor, semi-automatic feeders and nipple drinkers, with free access to diet and water.The minimum and maximum temperatures in the nursery room were 22.9 ± 1.50 °C and 31.8 ± 2.54 °C, respectively.
Pigs were fed in a two-phase feeding regimen (phase 1: 21-32 days, and phase 2: 32-45 days).All diets were corn and soybean meal-based with industrial amino acids and formulated according to the nutritional recommendations of the Brazilian Tables for Poultry and Swine 48 (Table 5), and provided in mash form.During phases 1 and 2, the dietary treatments consisted, respectively, of: (1) CON: control, basal diet, (2) NUC: CON + 1 g/kg and 0.5 g/kg of nucleotides, (3) YSC: CON + 20 g/kg and 10 g/kg of lysed yeast S. cerevisiae, (4) ASB: CON + 1.5 g/kg and 1 g/kg of acidifier sodium butyrate.Feed additives were added in replacement with inert in the CON based on the manufacturer's recommendations.

Growth performance and diarrhea incidence
Throughout the trial, the offered diet and leftovers were weighed to calculate average daily feed intake (ADFI).Pigs were individually weighed on d 21, 32, and 45 to determine BW, average daily weight gain (ADG), and feed conversion (FC).The fecal consistency of each pig was visually assessed during phase 1 (24-32 days) and phase 2 (32-45 days), using the method described by Liu et al. 49 .Fresh feces were ranked on a 4-point scale as follows: 0 = solid, 1 = semi-solid, 2 = semi-liquid, and 3 = liquid.The diarrhea incidence was defined as the consistency of feces at scale 2 or 3 for 2 continuous days.The diarrhea incidence for the pigs was calculated as follows: diarrhea www.nature.com/scientificreports/incidence (%) = [(the number of pigs with diarrhea in each pen × number of days of diarrhea) ÷ (total number of pigs in each pen × number of days)] × 100%.Observations were made in the morning, every day throughout the experimental period by a trained evaluator.The same blood donor piglet was electrically stunned (240 V for 3 s) followed by exsanguination to collect samples on d 32.Fragments measuring 2 cm were sampled (8 pigs/treatment) from the duodenum (10 cm from the pylorus junction), jejunum (mid-section), and ileum (5 cm to ileocecal junction) for histological evaluation 50 .The histological sections were then washed in a physiological solution (0.9% sodium chloride) and fixed in 4% paraformaldehyde solution (100 mL 40% paraformaldehyde, 900 mL distilled water, 2.28 g monobasic sodium phosphate, and 21.74 g dibasic sodium phosphate) for 24 h at room temperature 6 .Another 2 cm of jejunum was collected and immediately frozen in liquid nitrogen, stored at − 80 °C for RNA extraction and gene expression analysis.

Intestinal morphology, Peyer's patches, and goblet cells
After 24 h of fixation, the fragments of the duodenum, jejunum, and ileum were transferred to an ethanol solution 70% (v/v).Then, the samples were cut into cross-sections and dried in increasing gradients of ethyl, diaphanized in HistoChoice®, and embedded in liquid Paraplast® at 65 °C.Five cross-sections (5 μm thickness each) were placed per slide and stained with hematoxylin and eosin.The sections were semi-serial using 1 in 10 cuts 51 .For morphological readings of villus height and crypt depth in the duodenum, jejunum, and ileum, an EVOS™ M5000 Cell Imaging System optical microscope (Invitrogen, Thermo Fisher Scientific) with a 10-objective lens was used.The images were then analyzed using ImageJ 1.50i (Java1.6.0_20;National Institutes of Health, USA).Heights of 20 villus and their 20 crypts were selected and measured.Villus to crypt ratios using the length data were then calculated.All measurements were made by a single trained individual.In the ileum fragment, the total count of the Peyer's patches was performed at 4 × magnification 52 .
To assess the goblet cells in the duodenum, jejunum, and ileum, 10 fields per slide were photographed at 20 × magnification.Subsequently, the ImageJ program was used, and perpendicular lines were inserted with markings in uniformly sized quadrants under each image.Then, the total number of intersections in the image and the cells that touched the intersections were counted.The calculation was made according to the methodology proposed by Mandarim de Lacerda 53 :

Statistical procedures
The pen was considered the experimental unit for growth performance and diarrhea incidence analysis.One pig from each pen was considered the experimental unit for intestinal morphology, gene expression, and blood profile.The statistical model included the fixed effect of dietary treatment, and block and residual error as random factors.The normality of experimental errors was evaluated using Shapiro-Wilk.The data were analyzed using the mixed procedure of SAS 9.4 (SAS Inst., Inc., Cary, NC, USA) via one-way analysis of variance (ANOVA).When an effect was detected in the ANOVA (P < 0.05), differences between means were determined by the preplanned contrasts, in which each of the treatments (NUC, YSC, and ASB) were compared versus the control treatment (CON).The statistical significance and tendency were declared at P < 0.05 and 0.05 ≤ P < 0.10, respectively.

Goblet cells (%) =
total number of goblet cells × 100 total number total number of intersections

Table 3 .
Blood profile of piglets fed diets supplemented or not with feed additives on d 32 (n = 9 pigs replicates per dietary treatment).
a Dietary treatment: CON control, basal diet, NUC CON + 1 g/kg of nucleotides, YSC CON + 20 g/kg of lysed yeast S. cerevisia, ASB CON + 1.5 g/kg of acidifier sodium butyrate.b Pooled standard error of the mean.

Table 4 .
Intestinal morphology of piglets fed diets supplemented or not with feed additives on d 32 (n = 9 pigs replicates per dietary treatment).aDietary treatment: CON control, basal diet, NUC CON + 1 g/kg of nucleotides, YSC CON + 20 g/kg of lysed yeast S. cerevisiae, ASB CON + 1.5 g/kg of acidifier sodium butyrate.bPooled standard error of the mean.Item Dietary